Figure 3

VAI transcription is higher than U6 snRNA transcription in HeLa, DU145, and MCF-7 cells. The empty pGL3 vector (300 ng), as well as increasing concentrations of pGL3-U6 (100 ng, 200 ng, 300 ng) and pGL3-VAI (100 ng, 200 ng, 300 ng) were transiently transfected into asynchronous (A) HeLa, (B) DU145, and (C) MCF-7 cells. All luciferase assay results are expressed as relative light units (RLU): the average of the Photinus pyralis firefly activity observed divided by the average of the activity recorded from the Renilla luciferase vector. Experiments were done in triplicate, repeated three times, and representative experiments are depicted. (D) Western blot analysis of Brf1 and Brf2 protein levels in HeLa, MCF-7 and DU145 cells. 25 μg of nuclear extract was immunoblotted with anti-Brf1 (CS1043) and anti-Brf2 (CS1228) antibodies. As loading control, membrane was also immunoblotted with an anti-actin antibody, bottom panel. Arrows depict migration of actin, Brf1 and Brf2.